A better understanding of the molecular basis of both DNA double strand break (DSB) repair, and the immediate responses of human breast cancer cells to DNA damage after ionizing radiation (IR), which are important determinants in resistance, cell death or survival, will be required to improve therapy of advanced disease. Recently, the principal investigator's laboratory has isolated X-ray-inducible transcript-8 (xip8) [i.e., testosterone repressed message-2 (TRPM-2)/apolipoprotein J (ApoJ)], a protein that strongly associate with the Ku780 DSB repair protein. It is hypothesized that the induction and overexpression of the nuclear form of XIP8/TRPM-2/ApoJ after IR inhibits Ku70/Ku8O DNA end-binding, ATPase and/or helicase functions, resulting in loss of DNA- PK activity and nonhomologous DSB repair. XIP8 is a key determinant in apoptosis/survival responses in severely damaged human breast cancer (e.g., MCF-7) cells. The applicant will test this hypothesis by: (a) examining repair apoptotic and survival responses of MCF-7 cells that overexpress the nuclear form of XIP8, or in cells that are deficient in its expression (using cells from XIP8/ApoJ knockout mice) before and after IR (Years 0-5); (b) examining the effects of the nuclear form of XIP8 on Ku70-Ku-80 protein-protein interactions and DSB repair (Years 0-5); (c) elucidating the protein-protein interaction domains between XIP8 and Ku70 using yeast two-hybrid and co-immunoprecipitation analysis (Years 0-3); and (d) performing structure/function analysis of XIP8 in cells and cell lines from ApoJ/XIP8 knockout mice using Ku70/Ku8O activity assays and Ku70-XIP8 protein-protein interactions (Years 0-3). Dominant-negative versions of XIP8 and Ku70 will be constructed-for use as molecular radiosensitizers (Years 2-5).
These Specific Aims will directly test the hypothesis that the nuclear form of XIP8 is a determining factor in blocking nonhomologous DSB repair and regulating apoptosis when over-expressed in the nucleus of damaged cells. We will also determine if XIP8 induction plays a prominent role in cell cycle checkpoint regulation after IR.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA078530-04
Application #
6174057
Study Section
Radiation Study Section (RAD)
Program Officer
Pelroy, Richard
Project Start
1998-09-18
Project End
2002-08-31
Budget Start
2000-09-01
Budget End
2001-08-31
Support Year
4
Fiscal Year
2000
Total Cost
$213,201
Indirect Cost
Name
Case Western Reserve University
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Klokov, D; Leskov, K; Araki, S et al. (2013) Low dose IR-induced IGF-1-sCLU expression: a p53-repressed expression cascade that interferes with TGF?1 signaling to confer a pro-survival bystander effect. Oncogene 32:479-90
Leskov, Konstantin S; Araki, Shinako; Lavik, John-Paul et al. (2011) CRM1 protein-mediated regulation of nuclear clusterin (nCLU), an ionizing radiation-stimulated, Bax-dependent pro-death factor. J Biol Chem 286:40083-90
Markopoulou, Soultana; Kontargiris, Evangelos; Batsi, Christina et al. (2009) Vanadium-induced apoptosis of HaCaT cells is mediated by c-fos and involves nuclear accumulation of clusterin. FEBS J 276:3784-99
Pucci, Sabina; Mazzarelli, Paola; Paola, Mazzarelli et al. (2009) Interleukin-6 affects cell death escaping mechanisms acting on Bax-Ku70-Clusterin interactions in human colon cancer progression. Cell Cycle 8:473-81
Trougakos, Ioannis P; Lourda, Magda; Antonelou, Marianna H et al. (2009) Intracellular clusterin inhibits mitochondrial apoptosis by suppressing p53-activating stress signals and stabilizing the cytosolic Ku70-Bax protein complex. Clin Cancer Res 15:48-59
Trougakos, Ioannis P; Djeu, Julie Y; Gonos, Efstathios S et al. (2009) Advances and challenges in basic and translational research on clusterin. Cancer Res 69:403-6
Sawada, Motoshi; Sun, Weiyong; Hayes, Paulette et al. (2003) Ku70 suppresses the apoptotic translocation of Bax to mitochondria. Nat Cell Biol 5:320-9
Leskov, Konstantin S; Klokov, Dmitry Y; Li, Jing et al. (2003) Synthesis and functional analyses of nuclear clusterin, a cell death protein. J Biol Chem 278:11590-600
Yang, C R; Leskov, K; Hosley-Eberlein, K et al. (2000) Nuclear clusterin/XIP8, an x-ray-induced Ku70-binding protein that signals cell death. Proc Natl Acad Sci U S A 97:5907-12
Yang, C R; Wilson-Van Patten, C; Planchon, S M et al. (2000) Coordinate modulation of Sp1, NF-kappa B, and p53 in confluent human malignant melanoma cells after ionizing radiation. FASEB J 14:379-90

Showing the most recent 10 out of 11 publications